4.2.1: Instructions for using the UV-Vis Spectrophotometers

Below are the instructions for using the Agilent Cary Series UV-Visible spectrophotometer in the Absorption Spectrum of Conjugated Dyes experiment.

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 A. Start-up:

Determine which model of UV-vis instrument you are using.  Then, confirm that the UV-vis is switched "on". 

Agilent Cary 60	Agilent Cary 100
The Cary 60 instrument has a xenon flash lamp, which does not require warm up.	The Cary 100 instruments have tungsten and deuterium lamps that must be warmed up for approximately 1 hour prior to use for best results. However, since we are only doing qualitative analysis, it is sufficient for this experiment to warm them up for only a couple of minutes.
How to turn on the Cary 60: The Cary 60 should not be turned off. But, if it is off, check that the cell holders are empty and press the power button on the instrument.	How to turn on the Cary 100: With both sample and reference holders empty and the cover closed, turn on the instrument with the switch at the front, lower left. Wait two minutes before proceeding further.
Please leave this Instrument ON when you are finished with your experiment.	Always power OFF the Cary 100 when you are done for the day!

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B. Validate the Wavelength Accuracy 

1. Open the Validation Software by clicking the Cary WinUV shortcut on the desktop and selecting the Validate application. After a few moments the software screen will appear and will begin to communicate with the instrument. You can follow the progress by monitoring the display at the lower left of the screen. When the traffic light  in the upper center turns green, the system is ready. You will see absorbance in the upper left and wavelength in the upper right.
2.  Open the Validation method used for this course: go to File \(\rightarrow\) Open Method, navigate to the directory C:\CaryUVvis\Methods, and open the CHEM301L_Validation.MVD method file.

Figure \(\PageIndex{1}\): The Validate Main Screen. (CC-NC-BY-DUKE CHEM)

2. Click the button to view the Instrument Performance Tests. Discuss with your instructor which test(s) would be appropriate.  While you could run all of the tests listed, the type of test that is required is a Wavelength Accuracy test. There are several options for performing a wavelength accuracy validation. Depending on whether you are using a Cary 100 or a Cary 60, this test can be conducted using either the deuterium (D2) or xenon (Xe) lamp, respectively. Another good option is to use a holmium oxide (Ho₂O₃) standard if one happens to be on hand. Be sure to state in your notebook which instrument you are using and which specific test is used for the wavelength validation.
   Using the button, you can remove all tests from the list except for the Wavelength Accuracy test that you choose to use. The screen should look as shown here, with only the selected text in the list.

Figure \(\PageIndex{2}\): Selecting a Wavelength accuracy test. (CC-NC-BY-DUKE CHEM)

3. Click the button and, when the setup screen appears, place your names in the Name line; and, the name of the test in the comments box. In the Options section select Auto Print, Results and Graph. Data Form and Company Logo should not be checked. % Page Height set at 50 is fine.
4. Click to return to the main screen.
5. Check the sample compartment to be sure that there are no samples in the instrument and that there is nothing blacking the light path. Gently close the sample compartment.
6. Click Start   to begin the test. As the test proceeds the spectrum (or spectra) will be displayed. At the conclusion of the test, a report will be generated on the screen. Include these data in your laboratory notebook. Check to be sure that the difference between the expected and observed peak positions is one the order of \(\pm 0.2-0.4\) nm. If it is greater than that, consult your Instructor before proceeding further.

Figure \(\PageIndex{3}\): Results of the a Wavelength Accuracy Test using the Deuterium lamp emission on a Cary 100 series spectrophotometer. (CC-NC-BY-DUKE CHEM)

7. Exit the Validation software and return to the Windows Desktop.

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C. Setup Scan Application to Collect Absorption Spectra 

1. Now, from the Cary WinUV list of applications, select the Scan application. In a few moments the scan software main screen will appear and will begin to communicate with the instrument. You can follow the progress by monitoring the display at the lower left of the screen. When the traffic light at the top center of the screen turns green, the system is ready. You will see absorbance at the upper left and wavelength at the upper right of the screen.

Figure \(\PageIndex{4}\): The Scan Module Main Screen. (CC-NC-BY-DUKE CHEM)

2. Click the button and wait for the setup screen to appear. Note that the arrangement op options in the setup window depends on which instrument you are using (see Figure below). Begin on the Cary tab and in the Cary Instrument Control Options, set your scan range from 750 nm to 450 nm. In the Scan Controls, set the Ave time to 0.1 seconds and the Data interval to 1 nm (on the Cary 60, this is the same as the Medium setting). This will yield a Scan Rate of 600 nm/min. 

Figure \(\PageIndex{5}\): The Scan Setup Widnow, Cary Tab for Cary60 (left) and Cary100 (right) spectrometers. (CC-NC-BY-DUKE CHEM)

3.  The default settings are sufficient for most other options.  Click on each tab to check that the settings make sense.  Particularly for the Baseline Tab, None should be selected under Baseline Selection settings.

Figure \(\PageIndex{6}\): The Baseline Tab of the Scan Setup Screen. (CC-NC-BY-DUKE CHEM)

4. Finally, select the Reports tab.
   • Put your Section NUmber and Team Number in the Name box.
   • Put the Name of your Experiment, and the names of all team members in the Comment box.
   • Under Peak Table, put a check in All peaks.
   • Be sure the Include XY pairs table is not checked.
   • Click the Peak Information button, and set the Threshold to 0.200. Put Peaks in the Peak Type box.
   • Select for ASCII (csv) in the Autoconvert Option.
   • Click to close the Peak information box, and again to return to the main screen.

Figure \(\PageIndex{7}\): The Reports Tab of the Scan Setup Screen. (CC-NC-BY-DUKE CHEM)

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 D. Collect Absorption Spectra 

1. Place your first sample in the sample compartment and gently close the cover.
2. Click to begin the run.
3. You will be prompted for a filename.
   • Always name your files  using the convention YYYYMMDD_Team##_X.  Where YYYY is the four-digit year, MM is the two digit number of the month, DD is the two digit day of the month, Team## is your section and team number, and X is an indicator, like Dyes.  Since you will be running many samples, give the file the general name appended with a number "_1".  For example, 20260126_Team11_Dyes_01. 
   • Save your files in the directory C:\CaryUVvis\Data\current_year\course_semester.  
     For example, save in the folder C:\CaryUVvis\data\2026\CHEM301L_Spring26 if it is Spring 2026 and you are in CHEM301L.
4. You will then be prompted for a sample name. Place the name of the sample (Dye 1, two drops) in this box. 
5. As each run proceeds you will see the spectrum generated in real time on the screen. Make sure the maximum absorbance of each sample is between approximately 0.2 and 1.0; adjust the dye concentration to get the absorbance in this range.
6. At the conclusion of each run, place the next sample in the sample compartment and initiate the new scan. 
   *Note: the software prompts you to enter a filename and/or sample information BEFORE it scans that sample.  

Note

To add or remove traces from the spectrum view, click the button at the upper left of the toolbar, next to the Setup button. Check files to add them or uncheck to remove from the view. Then click Apply or OK. 

Figure \(\PageIndex{8}\): The Absorption Spectra of the Dyes. (Kathryn Haas; CC-NC-BY-SA)